The structure and function of the cell membrane, as well as many aspects of energy metabolism and cell cycling, are directly dependent on acyl-CoA thioesters and their CoA and fatty acid constituents. The complex interplay between CoA, short or long chain carboxylates, and acyl-CoA thioesters (and lipid derived products) in directing the functions and responses of cells is not well understood even at the most elementary level. This research program focuses on the delineation of cellular processes in which transformations of acyl-CoA metabolites or acyl-S-proteins play important roles. The catalysts for these transformations reside mainly in the hotdog fold superfamily. An integrated approach that combines the efforts of a diverse research team will be used to characterize hotdog thioesterases at the cellular, chemical, mechanistic, and structural levels. The overall goal is to gain an understanding of the relationships that exist between enzyme structures and the mechanisms of substrate recognition and catalysis in vitro and in vivo. This fundamental knowledge is required for the development of new drugs and for the discovery of new biochemical processes. An understanding of the relationship between in vitro chemical function (i.e., range of substrates and catalytic efficiencies) and the actual cellular chemical function (i.e., physiological substrates and in vivo catalytic efficiencies) will provide insight into the need for and mechanism of regulation of thioesterase activity within the cell. In addition, knowledge about relationships between structure and chemical and cellular functions should reveal how thioesterases successfully execute their evolved biological roles. PUBLIC HEALTH RELEVANCE: This project employs an integrated approach that relies on a multidisciplinary research team to define the hotdog thioesterases at the cellular, chemical, mechanistic, and structural levels. The results of this effort will facilitate the development of new therapeutic drugs and the discovery of new biochemical processes.